CN103667195B - 以nlrp3为靶点的药物筛选细胞模型及其应用 - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种以NLRP3为靶点的药物筛选细胞模型及其应用，该细胞模型是以哺乳动物细胞为宿主，转染入含NLRP33’-UTR核心序列与报告基因的重组质粒，所述NLRP33’-UTR核心序列为SEQ？ID1-9之一。本发明通过3’-UTR片段削减法寻找到能替代NLRP3mRNA全长的最小3’-UTR序列，将该序列与报告基因连接，然后将连接后的重组质粒转染HCT116细胞株形成稳定表达细胞株，随后加入待筛选的内源性代谢产物与天然化合物，测定报告基因的表达水平，同时设立阳性与阴性对照组，以此评价待筛选物质的活性，是一种灵敏、有效，并且能够适用于高通量抗感染药物的天然创新药物的筛选方法。
Description
技术领域
本发明涉及一种利用NLRP3基因3’-UTR稳定性建立的筛选模型来发现内源性稳定NLRP3mRNA的代谢产物，筛选外源性抗感染天然药物的新方法，属于分子药理学领域。
背景技术
NLRP3炎性体具有调控机体慢性炎症反应的功能，其组成包括NLRP3(NLRfamily，pyrindomaincontaining3)、凋亡相关斑点样蛋白(Apoptosis-associatedspeck-likeproteincontainingCARD，ASC)和半胱氨酸天冬氨酸蛋白酶-1(Cysteine-requiringaspartateprotease-1，Caspase-1)，是内源性或外源性危险信号的胞质内感受器，是活化caspase-1的分子平台，调控白细胞介素-1β(Interleukin-1β，IL-1β)和IL-18等促炎细胞因子的成熟和分泌。IL-1β是一个经典的促炎性细胞因子，活化的IL-1β与靶细胞上的IL-1受体结合，激活IL-1信号通路和髓样分化因子(Myeloiddifferentiationfactor，MyD88)依赖的NF-κB通路，促进IL-1等促炎因子转录，诱导机体炎症反应。研究证实，IL-1β是促进多种代谢性疾病发生发展的重要炎症因子，阻断其生物学效应能有效缓解代谢性疾病的进展。研究发现，在2型糖尿病(Type2diabetes，T2D)、动脉粥样硬化(Atherosclerosis，As)、非酒精性脂肪性肝炎(Non-alcoholicsteatohepatitis，NASH)、肥胖病、痛风和阿尔茨海默病(Alzheimer'sdisease，AD)中，相应的胰岛淀粉样多肽(IIAPP)、胆固醇晶体、棕榈酸盐、神经酰胺、尿酸盐晶体和β淀粉样蛋白(Aβ)均可通过激活NLRP3炎性体促进IL-1β成熟与分泌，提示NLRP3炎性体活化在机体异常代谢促进代谢性疾病进展中发挥关键作用。
NLRP3炎性体能够感知多种微生物及代谢产物激活信号，如MSU、焦磷酸钙晶体(Calciumpyrophosphatedihydrate，CPPD)、二氧化硅(Silica)、日本脑炎病毒(JapaneseEncephalitisvirus，JEV)、棕榈酸盐(Palmitate)、人呼吸道合胞病毒(Humanrespiratorysyncytialvirus，RSV)、β淀粉样蛋白(Amyloid-β，Aβ)、二氧化硅晶体(Silicacrystals)、胆固醇晶体(Cholesterolcrystals)等，不同信号激活NLRP3炎性体的机制不同。目前主要有以下3种假说：线粒体DNA假说、活性氧假说和溶酶体破裂假说。
第一种线粒体DNA假说。线粒体损伤释放的线粒体DNA(mitochondrialDNA，mtDNA)通过与NLRP3结合可以激活NLRP3炎性体。第二种活性氧假说。线粒体来源的活性氧(Reactiveoxygenspecies，ROS)是调控NLRP3炎性体活化的关键信号。第三种溶酶体破裂假说。细胞吞噬晶体或颗粒等物质后导致溶酶体酸化，肿胀破裂释放内容物至胞浆，激活NLRP3炎性体，其中组织蛋白酶-B(cathepsinB)是NLRP3炎性体活化最重要的上游信号。
病原体的刺激激活了NLRP3炎性体复合物的组装，进而启动了caspase-1的活化。活化的caspase-1能发挥多种生物学效应，这使得炎性体复合物在启动炎症反应和抗感染中发挥重要作用。
经由炎性体复合物活化的caspase-1作为一种酶，可以分解多种底物，包括前体IL-1、IL-18和IL-33等，使之成为活化形式。其中IL-1具有广泛的免疫调节作用，并具有致热和介导炎症的作用，它的生物学功能是通过与相应的高亲和力受体结合而介导的。IL-1通过与IL-1受体结合可以:①诱导单核/巨噬细胞产生IL-6和TNF，并通过诱导单核细胞和巨噬细胞产生IL-8介导对中性粒细胞的趋化作用；刺激中性粒细胞释放炎症介质，直接参与炎症发生过程，抑制粒细胞凋亡，促进粒细胞粘附和迁移。②促进胸腺细胞、T细胞的活化增殖和分化。③协同IL-4等细胞因子刺激B细胞的增殖和分化，促进免疫球蛋白的合成和分泌。④增强NK细胞的杀伤活性等。
NLRP3炎性体复合物在抗感染方面所起的重要作用正逐渐被研究所揭示。研究表明炎性体复合物通过活化caspase-1在抵御感染和清除病原微生物过程中起着非常重要的作用。caspase-1基因缺失的小鼠易于感染大肠杆菌、福氏志贺菌、鼠伤寒沙门氏菌和土拉热杆菌，而且抵御李斯特假单胞菌和白色念珠菌的能力也部分受损。在一项用腹膜内注射活的大肠杆菌造成的脓毒症动物模型实验中，caspase-1基因被敲除的小鼠更容易死于感染，其半数致死量比野生型杂合子小鼠低了3-4倍。相似地，用caspase-1抑制物Ac-YVADCHO处理野生型小鼠后，它们比未经处理过的小鼠更易于感染大肠杆菌。注射重组小鼠IL-1β和IL-18后，caspase-1敲除小鼠重新获得了抵抗感染的能力。鼻内感染福氏志贺菌可以致死caspase-1敲除小鼠，但不会引起野生对照组小鼠的死亡。caspase-1缺失可以加重感染，使感染后24h和48h的病原菌负荷量较野生对照组分别增加10倍和670倍。与大肠杆菌和福氏志贺菌相同，caspase-1敲除小鼠表现对经口感染鼠伤寒沙门氏杆菌有更高的易感性，死亡时间较对照组明显缩短，且在派氏淋巴结区、肠系膜淋巴结和脾脏里的菌量明显增多。在腹膜内感染鼠伤寒沙门氏菌的caspase-1基因敲除小鼠脾脏和肝脏内也展现出了细菌负荷量的增高。相似的情况也见于土弗拉热菌、李斯特假单胞菌等感染。
脓毒症是危重症医学中较为常见的一种综合征，它被定义为因病原体感染而引起的全身炎症反应综合征。鉴于NLRP3炎性体复合物及其活化的caspase-1在启动炎症反应和抗感染方面等方面的作用，有研究者试图去探索NLRP3炎性体复合物与脓毒症之间的联系。Stefen等研究发现，用肺炎支原体感染NLRP3基因缺失的老鼠，结果显示试验组老鼠肺部炎症较对照组更为严重，并且低存活率更低，表明NLRP3基因的整体保护作用。Andrea等发现，在小鼠脓毒症模型中，caspase-1、ASC基因敲除的小鼠脾脏和肝脏细胞感染程度明显高于对照组，这表明ASC调节的炎性反应在免疫反应中的重要作用。Fahy等通过比较脓毒症休克、ICU重症非脓毒症患者、正常对照的三组外周血单核细胞(PBMC)中Toll样受体、NLR家族蛋白、细胞因子和NF-κB相关基因等的mRNA表达发现，脓毒症休克组NLRP1炎性体复合物成分ASC、caspase-1和NLRP1的mRNA水平显著低于重症非脓毒症患者组，NLRP1的mRNA水平与脓毒症患者的生存率具有相关性(P＝0.0068)，且与简化急性生理Ⅱ(SAPSⅡ)评分呈负相关(r＝-0.63)Giamarellos-Bourboulis等通过研究炎性体复合物活性发现，脓毒症患者外周血单个核细胞(PBMC)中pro-caspase-1和活化的caspase-1水平均明显降低；脓毒症患者PBMC产生IL-1β的能力受损，且阻断caspase-1后IL-1β的释放明显受到抑制。因此NLRP3炎性体复合物以及caspase-1表达和活化的减少及IL-1β释放受抑可能是导致脓毒症早期免疫麻痹的重要原因。综上所述，NLRP3炎性体复合物及其活化的caspase-1在介导脓毒症全身炎症反应和抗感染方面均发挥着重要作用，因此在保证有效的抗感染能力的前提下使炎症反应限制在适度范围内将对脓毒症的预后产生有利的影响，炎性体复合物及其活化的caspase-1等可能成为有效的调节靶点。
值得注意的是，鉴于NLRP3在机体炎症与抗感染方面的重要性，寻找更多内源性NLRP3激活代谢产物有助于深入理解NLRP3的病理生理意义；同时以NLRP3为靶点寻找能增强NLRP3活性的天然物质，对脓毒症等机体抗感染能力低下的疾病有重要的应用价值。但遗憾的是，到目前为止人们对NLRP3基因表达调控，尤其是发生在转录后水平的调控机制的认识尚处于未知状态。实际上，转录后水平的调控具有快速、经济等特点，相对于转录水平的调节具有更好的灵活性与机体适应性。
发明内容
为了解决现有技术存在的问题，本发明提供了一种以NLRP3为靶点的药物筛选细胞模型，及其应用。
本发明以NLRP3为靶点的药物筛选细胞模型，是以哺乳动物细胞为宿主，其中转染有含NLRP33’-UTR核心序列与报告基因的重组质粒。所述NLRP33’-UTR核心序列为SEQID1-9之一；优选SEQID9。
本发明还公开了上述以NLRP3为靶点的药物筛选细胞模型的应用，即该模型在筛选内外源性NLRP3激活剂中的应用。具体方法是在细胞模型中加入待筛选的样品，测定报告基因的表达水平，评估内外源性NLRP3激活剂的生物活性；将得到的活性高的内源性激活性进入深入研究其体内作用机制，外源性激活剂进行抗感染的药理实验，作为抗感染的候选药物。
本发明中所述内外源性NLRP3激活剂包括哺乳动物细胞体内代谢产物，各种小分子化合物库，中药与西药有效成分或有效部位。
本发明所述以NLRP3为靶点的药物筛选细胞模型，是通过下述方法获得：通过分子克隆与3’-UTR片段削减法寻找到能替代NLRP3mRNA全长的3’-UTR核心序列；将NLRP33’-UTR核心序列与报告基因连接得重组质粒；并将该重组质粒转染入哺乳动物细胞，构建并筛选出稳定表达细胞模型。
本发明首次发现调控NLRP3mRNA稳定性的核心序列，并将此序列构建成报告基因表达质粒，构建稳定表达细胞株，用于筛选内外源性NLRP3激活剂，具有明显的创新价值与应用前景。
附图说明
图1：pmirGLO示意图。
图2：NLRP33'UTR报告基因转染结果。
图3：NLRP33'UTR全长以及变体V1和V2报告基因转染结果。
图4：NLRP33'UTR全长以及变体V4.1、V4.2、V4.3和V4.4报告基因转染结果。
图5：NLRP33'UTR全长以及V4.4.1和V4.4.2变体报告基因转染结果。
图6：NLRP33'UTRV4.4.2变体稳定表达细胞筛选体系用于药物筛选。
图7：黄芩苷的化学结构。
具体实施方式
1材料与方法
1.1实验方法
Dual-LuciferaseReporterAssaySystem购自Promega。XbaI、SacI限制性内切酶、PrimerSTARHSDNAPolymerase、T4DNALigase购自TaKaRa
TaqDNAPolymerase购自TIANGEN，DMEM购自HyClone。
1.2实验方法
1.2.1克隆构建
(1)首先用PCR方法从基因组中或从质粒中扩增得到目的片段，电泳，胶回收目的片段。
(2)酶切目的片段和质粒载体
(3)制备top10感受态(无菌操作)
①接45μltop10过夜菌于一LB管中(有3ml),37℃摇床1h，至OD值0.15-0.25之间(勿过)。
②分装2个无菌EP管中，离心，4℃，3400rpm，5min。
③弃上清，加700μl0.1MCaCl2溶液，冰上30min。
④离心，4℃，3400rpm，5min。
⑤弃上清，加100μl的CaCl2溶液，冰上2h-24h内能用。也可加15μl保种甘油保存于-70℃。
(4)酶切后胶回收
①将酶切后的片段和质粒1％Agarose胶电泳，同时用未酶切载体作对照
②在紫外光下将含目的片段DNA的胶切下至1.5mleppendorf管中。
③加500μlSolutionN，55℃溶解10min。
④加50μlSolutionB，55℃，1min。
⑤将液体转移至3S柱内，室温2min，离心12000rpm，1min。
⑥倒去管内液体，加700μlWashingsolution，离心12000rpm，30s。
⑦重复(6)一次。
⑧倒去管内液体，甩空12000rpm，2min。
⑨将3s柱放入干净1.5mleppendorf管中，在膜中央加55℃预热过的DDW30μl，放置5min溶解。
⑩离心，12000rpm，2min。回收到干净1.5mleppendorf管中。
(6)连接
(7)连接产物的转入，冰上操作，无菌
①取20μl连接产物体系加入100μl感受态细胞，轻吹混匀，冰上孵育40min。
②准备42℃水浴，90s精确热休克。
③立即拿出置冰上3min。
④加700μlLB，轻轻混匀。
⑤将转的连接产物37℃水浴培养45min以上。
⑥拿出含氨苄的LB平板，于37℃温箱。
⑦离心，3500rpm，5min，涂板，最适体积：100-200μl。
⑧烧涂棒，使其冷却，吹入适量菌液，涂平，涂完烧涂棒。
⑨做好标记：日期、名字、内容。
⑩正置片刻，倒置于37℃温箱中培养12h左右。
(8)菌体PCR鉴定
①取出平板观察
②在连接板上用无菌枪头挑取多个菌落，打入事先准备好的加好20μlLB培养基的EP管，标记好
③取2μl作模板加入已分装好的PCR小管中，进行菌体PCR，剩余的放4℃冰箱暂存
④1％Agarose电泳PCR产物
(9)接种培养
①根据电泳结果，看有无阳性克隆，若有，准备相应个数的LB培养基玻璃管
②加1/1000抗生素(Amp)
③加10μlEP管内有菌的LB，做好标记
④37℃摇床培养14-16h
(10)小体质粒(试剂盒)
①离心收集菌体(约3ml，留一些保种)，4000rpm，5min
②加100μlSolutionI，先枪头吹散，再振荡器混匀，充分悬浮菌体，静置3-5min
③冰上操作，加200μlSolutionII，立即上下颠倒，冰浴2-3min至溶液变澄清
④加入400μlSolutionIII，立即上下颠倒使其充分中和，室温静置2-3min
⑤高速离心，12000rpm，10min
⑥将上清转移至3s柱内，室温2-15min，离心10000rpm，1min
⑦600μlWashingsolution洗两次
⑧空甩，10000rpm，2min
⑨更换管套，50μl预热过的DDW至膜中央，静置2min以上
⑩离心，10000rpm，2min收集液体于干净的EP管中
(11)酶切鉴定
酶切体系与步骤2中的相同，1％Agarose胶鉴定
(12)
将阳性结果送测序，如果结果正确就大提质粒
(13)碱法大量提取质粒DNA
1)菌液倒入500ml离心杯，注意二杯平衡，收集菌体，5000rpm，5min，15℃
2)用滴管加入10ml预冷的SolutionI，吹匀
3)加入20mlSolutionII，盖上盖子，轻轻上下颠倒，转至澄清
SolutionII需要临时配置：0.2MNaOH，1％SDS
4)加入15mlSolutionIII，离心8000rpm，10min，4℃，轻轻取出SolutionI、II、III的量可变，但要遵循2:3:4的比例
5)取出上清用棉花过滤，加入60％体积的异丙醇，室温放置5-30min。精确平衡，离心，常温20-25℃，8000rpm，10min
6)倒去上清液体，瓶子倒扣控干，收集沉淀。加3mlTE轻吹使其溶解(溶液应透明)。加入50ml离心管中，加3ml预冷的LiCl(等体积的)，放置5min。离心：4℃，12000rpm，10min7)收集上清加入等体积的预冷异丙醇，充分混匀，冰上放置5-10min，离心：20℃，12000rpm，10min
8)收集沉淀于EP管中，做标记，溶于50μlTE，加入50×RNase，15-20μl，37℃放置20-40min9)加入500PEG800，4℃沉淀半小时左右(也可过夜)。离心：10000rpm，10min，4℃
10)收集沉淀，加入400TE吹散溶解，加入等体积的苯酚抽提，漩涡仪上混匀。离心：4℃，12000rpm，2.5min
11)收集上层溶液，加入400μl异丙醇/氯仿混合液(1:24)抽提两次。离心：4℃，12000rpm，2.5min
12)收集上层溶液，加入100μl10N乙酸铵(或加1/10体积的醋酸钠，3M,PH5.2)，再加两倍体积预冷的乙醇。10000rpm，10min，4℃
13)收集沉淀，用70％乙醇洗一遍，无菌操作。离心：12000rpm，2min，超净台中置10-20min，晾干。
14)加200-500μlDDW(水浴温热)
15)测浓度：100μlDDW+1μlplasmid，设对照(DDW)，分光光度计用dsDNA档，记录数据：浓度、260/280、260/230
1.2.2细胞培养
培养基配制：DMEM+10％胎牛血清+1‰抗生素(青霉素+链霉素)，A549细胞置37℃，5％CO2温箱培育。
细胞传代：用滴管吸去旧培液，加5ml胰酶消化半分钟至看到有细胞掉落，加培养终止消化，吹匀，离心：4℃，1000rpm，3min。倒去上清，加5ml新培液吹散，加入培瓶继续培养。1.2.3质粒转染
铺细胞于24孔板中过夜，长至70-80％时转染。每个孔转染质粒0.8μg，1.6μlPolyjet。用不加血清的DMEM稀释质粒与转染试剂，分别稀释1.6μl转染试剂和0.8μg质粒于50μl无血清的DMEM中，混匀后把两者混合，室温放置15min后，加入细胞孔中。转染6-10小时后换液，转染24小时后收集细胞，测报告基因。
1.2.4Dual-luciferase(下述溶液体积对应于24孔24孔板的一个孔)
收24孔板细胞，加1×celllysisbuffer50μl,冰上裂解15min。高速离心：12000rpm，4℃，2min×①LuciferaseassayreagentII
将10mlLuciferaseassaybufferII加到LuciferaseAssaySubstrate瓶中，混匀，分装成20小管，500μl每管标明LARII字样。
②Stop&Glo试剂
将20μl的50Stop&Glosubstrate加到1ml的Stop&Globuffer中，混匀，分装500μl每管。以上两种底物使用时现配制，每块24孔板各取一小管，其余-70℃保存，配制和保存时均需注意避光。
(2)裂解细胞时可使用2mlEP管分装反应液I(LARII)，准备stop试剂。
(3)(打开)luminometer仪器，选择protocols→RunPromegaProtocol→DLR-0-1NJ→OK。
(4)吸取10μl蛋白液至事先分装好的10μl反应液I中，混匀(吹十下并将液体甩至管底)，用luminometer仪器读数(按键measureLuminescance),得目的值a，吸取10μlstop试剂加入(震10s并将液体甩至管底，按OK键)，终止前一个底物的反应并测内标值b。记录数据a、b及其比值a/b。
1.2.5数据处理和分析
所有数据均用mean±SEM表示。对于两组比较，用Student'sttest进行检验，P<0.05时有显著性差异，P<0.01时有及其显著性差异。
2、实验结果
2.1从人类基因组DNA中扩增NLRP33'-UTR
设计NLRP33‘UTRPCR扩增引物(引物序列如下Sense:ATATGAGCTCGAGTGGAAACGGGGCTGC(SEQID10)；Anti-sense:GCGCTCTAGAAAAATTAAGAAAAGGAATCAATTTAATTAAATACCAAAAAGTTTTAC(SEQID11)),使用PrimerSTARHSDNAPolymerase，从人类基因组DNA中PCR扩增NLRP33‘UTR，退火温度设置为60℃，30个循环。PCR产物电泳得到一条单一条带，大小与目的基因相符，胶回收PCR产物。分别用XbaI、SacI限制性内切酶酶切目的片段和pmirGLO载体(载体来自Promega公司)(图1)，连接并转化至感受态细胞，离心收集菌体涂布于含氨苄的平板上。挑取单克隆菌落进行菌液PCR和酶切鉴定，将鉴定结果为阳性的菌液送至测序公司测序。测序后与NCBI数据库比对正确，NLRP33'-UTR核酸序列如SEQID.1
2.2检测NLRP33'-UTR对报告基因表达的影响
将上述扩增获得的NLRP33'-UTR序列通过酶切，连接，构建进入pmirGLO报告载体，lipofectamine2000转染入HCT116细胞中，Dual-luciferase方法测定转入NLRP33'-UTR后报告基因活性。结果显示，插入NLRP33'UTR全长后，报告基因表达明显下降(如图2)，表明NLRP33'-UTR自身是不稳定的，3'-UTR上可能含有多种去稳定NLRP3mRNA的序列。
2.3构建NLRP33'-UTR变体V1和V2
为寻找决定NLRP3mRNA稳定性的最小3'UTR序列，以克隆出来的NLRP33'UTR为模板，分两段扩增NLRP33'UTR两个变体V1和V2。变体V1共254bp，变体V2共344bp，变体V1和V2序列如下SEQID2和SEQID3。分别用XbaI、SacI限制性内切酶酶切目的片段和pmirGLO载体，连接并转化至感受态细胞，离心收集菌体涂布于含氨苄的平板上。挑取单克隆菌落进行菌液PCR和酶切鉴定，将鉴定结果为阳性的菌液送至测序公司测序。测序后与NCBI数据库比对正确，
2.4检测并比较NLRP33'-UTR及变体V1和V2对报告基因表达的影响
将已经构建好的含NLRP33'UTR全长以及变体V1和V2报告质粒转入HCT116细胞中，Dual-luciferase方法测定转入NLRP33'-UTR后报告基因活性。结果显示，插入NLRP33'UTRV2变体后，报告基因表达明显下降，但下降幅度没有NLRP33'UTR全长明显，NLRP33'UTR主要是V2变体起到了降低报告基因稳定性的作用(如图3)。
2.5扩增NLRP33'UTR四个变体V4.1、V4.2、V4.3和V4.4
以克隆出来的NLRP33'UTR为模板，利用分子克隆方法将V1拆分为V4.1，V4.2两个变体；将V2拆分为V4.3，V4.4；变体V4.1共126bp(SEQID4)，变体V4.2共128bp(SEQID5)，变体V4.3共183bp(SEQID6)，变体V4.4共161bp(SEQID7)。分别用XbaI、SacI限制性内切酶酶切目的片段和pmirGLO载体，连接并转化至感受态细胞，离心收集菌体涂布于含氨苄的平板上。挑取单克隆菌落进行菌液PCR和酶切鉴定，将鉴定结果为阳性的菌液送至测序公司测序。测序后与NCBI数据库比对正确
2.6检测并比较NLRP33'UTR及变体V4.1、V4.2、V4.3和V4.4对报告基因表达的影响
将上述构建成功的含NLRP33'UTR全长以及V4.1、V4.2、V4.3和V4.4变体报告质粒转染入HCT116细胞中，运用Dual-luciferase方法测定转入NLRP33'UTR及其变体后报告基因后活性变化。结果如图4，NLRP33'UTR主要是V4.4变体起到了降低报告基因稳定性的作用。
2.7扩增NLRP33'UTR变体V4.4的两个变体V4.4.1和V4.4.2
进一步以克隆出来的NLRP33'UTRV4.4为模板，利用分子克隆方法进一步拆分为2个变体，V4.4.1共80bp(SEQID8)，变体V4.4.2共81bp(SEQID9)。分别用XbaI、SacI限制性内切酶酶切目的片段和pmirGLO载体，连接并转化至感受态细胞，离心收集菌体涂布于含氨苄的平板上。挑取单克隆菌落进行菌液PCR和酶切鉴定，将鉴定结果为阳性的菌液送至测序公司测序。测序后与NCBI数据库比对正确。
2.8检测并比较NLRP33'UTR及变体V4.4.1和V4.4.2对报告基因表达的影响
将上述已经构建好的含NLRP33'UTR全长以及V4.4.1和V4.4.2变体报告质粒染入HCT116细胞中，运用Dual-luciferase方法测定转入NLRP33'UTR及其变体后报告基因表达的变化。结果显示NLRP33'UTR主要是V4.4.2变体起到了降低报告基因稳定性的作用(如图5)。
2.9稳定表达NLRP33'-UTRV4.4.2变体的细胞株建立
在获取调节NLRP3mRNA稳定性的最小3’-UTR序列后，我们采用lipofectamine转染方法建立稳定表达NLRP33'-UTRV4.4.2的HCT116细胞株。首先摸索G418致死浓度为600ug/ml。细胞按1：10稀释后，改换含600不敷出μg/mlG418的完全培养液中进行培养、筛选。待未转染细胞全部死亡后，改用400μg/ml的G418维持筛选，每三天换液一次，去除坏死的细胞碎片。筛选2周后获得G418抗性克隆。同时转染空白载体作为阴性对照组及仅加入脂质体不加质粒的空白对照组。
2.10验证细胞株作为筛选模型的应用
将稳定转染NLRP33'-UTRV4.4.2变体的细胞接种于96孔上，加入已经的NLRP3激活剂与其它具有抗感染作用的小分子化合物，结果发现尿酸盐结晶(内源性NLRP3激活物质)与从中药黄芩中获得的黄芩苷具有明显的稳定NLRP33'-UTR的作用(图6)。多种实验表明中药黄芩具有很强的抗菌抗感染作用，对痢疾杆菌、伤寒杆菌、副伤寒杆菌、霍乱弧菌、大肠杆菌、变形杆菌、绿脓杆菌、葡萄球菌、溶血性琏球菌、肺炎双球菌、白喉杆菌等有抑制作用，而黄芩苷是黄芩的主要有效成分(图7)。以上结果表明该模型用于筛选NLRP3的激活剂是有可行性的。
Claims (5)
1.一种以NLRP3为靶点的药物筛选细胞模型，是以哺乳动物细胞为宿主，转染入含NLRP33’-UTR核心序列与报告基因的重组质粒，所述NLRP33’-UTR核心序列为SEQID2-9之一。
2.如权利要求1所述的细胞模型，其特征在于所述哺乳动物细胞为HCT116细胞。
3.如权利要求1所述的细胞模型，其特征在于报告基因为荧光素酶基因。
4.权利要求1所述以NLRP3为靶点的药物筛选细胞模型在筛选内外源性NLRP3激活剂中的应用。
5.如权利要求4所述的应用，其特征在于所述的内外源性NLRP3激活剂包括哺乳动物细胞体内代谢产物，各种小分子化合物库，中药与西药有效成分或有效部位。
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